Lactic acid-responsive high-efficiency anti-tumor chemotherapy-immunotherapy nano composite material and preparation method thereof

Abstract

The invention discloses a lactic acid-responsive nano composite material for antitumor chemotherapy-immunotherapy. The structure of the nano composite material is a nano structure formed by compounding PEGylated viral vaccine protein, lactate oxidase and hollow manganese dioxide loaded with a hypoxia-sensitive chemotherapeutic drug; the nano composite material provided by the invention can simultaneously induce a tumor to release a large amount of antigens, promote an antigen cell presentation process and improve the tolerance of the tumor to immunotherapy, can efficiently activate natural immune response of a tumor part, realizes a tumor inhibition effect, can regulate and control natural immunity of the tumor from multiple aspects, and has a wide application prospect. And safe and efficient anti-tumor chemotherapy-immunotherapy is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a lactic acid-responsive high-efficiency anti-tumor chemotherapy-immunotherapy nanocomposite material and a preparation method thereof. Background Art

[0002] Using chemotherapy drugs to induce the immunogenic death effect of tumor cells and synergizing it with immunotherapy is currently a popular method for treating malignant tumors, and major breakthroughs have been made in clinical practice.

[0003] The activation of innate immunity in the tumor site is crucial for the subsequent triggering of an efficient acquired immune response. However, the amount of antigens released by tumor cells under single chemotherapy stimulation is insufficient, making it difficult to effectively activate innate immune cells, and there are multiple immune tolerance mechanisms in tumor tissues. These factors have severely limited the application of chemotherapy-immunotherapy in the field of tumor treatment.

[0004] In view of this, some researchers have increased oxidative stress in tumor cells during chemotherapy to amplify the immunogenic death effect triggered by chemotherapy, but these studies cannot avoid the toxic side effects of drug off-target effects on the body. On the other hand, many reports have combined immunoadjuvants with chemotherapy to increase the activation of natural immunity through a cascade immune regulation strategy that induces tumor cells to release antigens and promotes antigen presentation by natural immune cells. However, these strategies have a relatively single pathway of action and cannot effectively overcome the immune tolerance of tumors.

[0005] Therefore, how to achieve safe and efficient chemotherapy-immunotherapy is crucial to promoting its application in clinical tumor treatment.

[0006] Designing a tumor-responsive activation therapeutic platform using substances overexpressed in tumor sites is an effective strategy to overcome the toxicity of traditional treatment systems. Lactic acid, as one of the substances accumulated in large quantities in tumor sites, can induce the upregulation of multiple immunosuppressive factors in tumors and enhance the tolerance of tumors to immunotherapy. It has become the focus of many tumor treatment strategies. Summary of the invention

[0007] The object of the present invention is to provide a lactic acid-responsive, highly efficient anti-tumor chemotherapy-immunotherapy nanocomposite material and a preparation method thereof, so as to solve the problems raised in the above-mentioned background technology.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a lactic acid-responsive anti-tumor chemotherapy-immunotherapy nanocomposite material, wherein: the structure of the nanocomposite material is a nanostructure formed by a composite of PEGylated viral vaccine protein, lactate oxidase and hollow manganese dioxide loaded with hypoxia-sensitive chemotherapy drugs.

[0009] Preferably, the hypoxia-sensitive chemotherapeutic drug is loaded into the inner cavity of the hollow manganese dioxide in the nanocomposite material.

[0010] Preferably, the diameter of the nanocomposite material is 150 nm - 200 nm.

[0011] Another object of the present invention is to provide a method for preparing a lactate-responsive anti-tumor chemo-immunotherapy nanocomposite material, which includes the following steps:

[0012] Step 1, preparing a positively charged hollow manganese dioxide nano-loading system: uniformly dispersing hollow manganese dioxide nanoparticles in a chemotherapeutic drug solution, washing by oscillation, and then compounding with a positively charged polymer solution to obtain a positively charged nano-loading system;

[0013] Step 2, loading lactate oxidase and virus vaccine protein: adding lactate oxidase to the nano-loading system, centrifuging after oscillation, adding PEGylated virus vaccine protein and mixing evenly, and forming a nanocomposite material by repeated extrusion.

[0014] Preferably, the PEGylated virus vaccine protein is an inactivated vaccine for canine coronavirus for veterinary use, the positively charged polymer solution is polyallylamine hydrochloride, and the chemotherapeutic drug is a hypoxia-responsive anti-tumor chemotherapeutic drug.

[0015] Preferably, the hypoxia-responsive anti-tumor chemotherapeutic drug is tirapazamine.

[0016] Preferably, Step 1 includes the following steps:

[0017] Step 1.1: Reacting a KMnO4 solution with an aqueous solution of sSiO2 nanoparticles under ultrasonic conditions, centrifuging to collect the product, and etching the sSiO2 component in the product with a Na2CO3 solution to obtain hMnO2 nanoparticles;

[0018] Step 1.2: Dispersing hMnO2 in a chemotherapeutic drug solution, oscillating, washing with water, and continuing to oscillate with a positively charged polymer solution, and centrifuging and washing to obtain drug-loaded nanoparticles.

[0019] Preferably, the mass ratio of KMnO4 to sSiO2 is 15:2, and the mass ratio of the positively charged polymer solution to hMnO2 is 2:1

[0020] Preferably, Step 2 is specifically: adding a lactate oxidase solution to the nano-loading system, oscillating, adding a vaccine protein solution, vortexing and mixing evenly, and repeatedly extruding the mixed solution with a liposome extruder.

[0021] Preferably, the feeding mass ratio of hollow manganese dioxide to lactate oxidase and vaccine protein is 10:1 and 1:1.

[0022] Technical effects and advantages of the present invention:

[0023] 1. The nanocomposite provided by the present invention can simultaneously induce tumors to release a large amount of antigens, promote the antigen presentation process by antigen-presenting cells, and improve the tolerance of tumors to immunotherapy. It can efficiently activate the innate immune response at the tumor site, achieve tumor suppression effects, and can regulate the innate immunity of tumors from multiple aspects to achieve safe and efficient anti-tumor chemo-immunotherapy;

[0024] 2. The nanocomposite provided by the present invention can respond to intratumoral lactic acid for chemotherapy and chemodynamic therapy. The raw materials are commercially available vaccines, lactate oxidase, and inorganic nanomaterials. Using commercially available vaccines as immune adjuvants, the cost is low, and by compounding with immune adjuvants with high biosafety, the innate immunity of tumors can be regulated from multiple aspects to achieve safe and efficient immune activation effects. Description of the drawings

[0025] Figure 1 a shows the morphological structure of the nanocomposite under a transmission electron microscope; Figure 1 b shows the hydrated particle size diagram of the nanocomposite; Figure 1 c shows the ultraviolet-visible absorption spectra of different intermediate products during the sample preparation process; Figure 1 d shows the polyacrylamide gel electrophoresis of the nanocomposite after degradation; Figure 1 e- Figure 1 f shows the effect diagram of the nanocomposite consuming lactic acid to produce H2O2; Figure 1 g shows the effect diagram of the nanocomposite catalyzing the production of reactive oxygen species; Figure 1 h shows the effect diagram of the nanocomposite consuming oxygen.

[0026] Figure 2 a- Figure 2 d shows the in vitro anti-tumor effects of different control materials and the nanocomposite loaded with TPZ.

[0027] Figure 3 The ability of the nanocomposite to induce immunogenic death of tumor cells: Figure 3 a shows the effect of the nanocomposite triggering the release of HMGB1 from tumor cells; Figure 3 b shows the ability of the nanocomposite to trigger the exposure of calreticulin on tumor cells.

[0028] Figure 4 The activation effect of the nanocomposite on antigen-presenting cells: Figure 4 a shows the activation effect of the nanocomposite on macrophages; Figure 4 b shows the activation effect of the nanocomposite on dendritic cells.

[0029] Figure 5In vivo immune activation effect of the nanocomposite after intravenous injection: Figure 5 a shows the effect of the nanocomposite in downregulating M2 macrophages in the tumor; Figure 5 b - 5c show the activation effect of the nanocomposite on macrophages and dendritic cells in the tumor; Figure 5 d - Figure 5 e shows the promoting effect of the nanocomposite on CD4 + T cells and CD8 + T cells.

[0030] Figure 6 Tumor enrichment ability and biosafety of the nanocomposite: Figure 6 a shows the enrichment of the nanocomposite at different time points in the tumor site after intravenous injection; Figure 6 b - 6c show the blood biochemical and blood routine analysis of mice after intravenous injection.

[0031] Figure 7 In vivo antitumor effect of the nanocomposite: Figure 7 a shows the growth curve of the tumor tissue of each mouse during the treatment; Figure 7 b shows the pathological sections of the tumor tissue after treatment.

[0032] Figure 8 Antitumor mechanism of the nanocomposite: Activate kinetic therapy and chemotherapy by oxidizing lactic acid in the tumor, reverse the immunosuppressive tumor microenvironment, and amplify the innate immune response of the tumor through an inactivated vaccine against canine coronavirus for veterinary use. Specific implementation mode

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] The present invention provides a lactate-responsive anti-tumor chemo-immunotherapy nanocomposite, wherein:

[0035] The structure of the nanocomposite is a nanostructure formed by the complexation of PEGylated virus vaccine protein, lactate oxidase, and hollow manganese dioxide loaded with hypoxia-sensitive chemotherapeutic drugs

[0036] The structure of the nanocomposite is a nanostructure formed by the complexation of PEGylated virus vaccine protein, lactate oxidase (LOX), and hollow manganese dioxide (hMnO2) loaded with hypoxia-sensitive chemotherapeutic drugs.

[0037] In the present invention, the nanocomposite has good tumor enrichment ability, is a composite material with an inhibitory effect on tumors, and is a composite material with high biosafety.

[0038] Specifically, the nanocomposite loads hypoxia-sensitive chemotherapeutic drugs through the lumen of hMnO2, composites the vaccine and enzyme protein components with hMnO2 nanoparticles carrying hypoxia drugs through physical extrusion and electrostatic interactions, activates chemotherapy and chemodynamic therapy by oxidizing intratumoral lactic acid, and promotes the release of a large amount of antigens by tumor cells; promotes the antigen presentation process through canine coronavirus vaccine protein; and the nanocomposite alleviates the immune tolerance of tumors by consuming lactic acid.

[0039] Specifically, the diameter of the nanocomposite is 150 nm - 200 nm, which is suitable for systemic intravenous administration.

[0040] Another object of the present invention is to provide a preparation method of a lactate-responsive anti-tumor chemotherapy-immunotherapy nanocomposite, which includes the following steps:

[0041] Step 1, prepare a positively charged hMnO2 nanoloading system: uniformly disperse hMnO2 nanoparticles in a chemotherapeutic drug solution, after oscillation and washing, composite with a positively charged polymer solution to obtain a positively charged nanoloading system;

[0042] Step 2, load LOX and virus vaccine protein: add LOX to the nanoloading system, after oscillation and centrifugation, add PEGylated virus vaccine protein and mix evenly, and form a nanocomposite through repeated extrusion, namely a lactate-responsive anti-tumor chemotherapy-immunotherapy nanocomposite.

[0043] Specifically, the PEGylated virus vaccine protein is a veterinary canine coronavirus inactivated vaccine, the positively charged polymer solution is polyallylamine hydrochloride (PAH), and the chemotherapeutic drug is a hypoxia-responsive tumor chemotherapy drug.

[0044] Specifically, the hypoxia-responsive tumor chemotherapy drug is tirapazamine (TPZ).

[0045] Specifically, Step 1 includes the following steps:

[0046] Step 1.1: React a 10 mg / mL KMnO4 solution with a 1 mg / mL sSiO2 nanoparticle aqueous solution under ultrasonic conditions for 6 h. After centrifuging (12,000 rpm, 15 min) to collect the product, etch the sSiO2 component in the product with a 2 M Na2CO3 solution to obtain hMnO2 nanoparticles;

[0047] Step 1.2: Disperse 10 mg of hMnO2 in a 5 mg / mL chemotherapeutic drug solution, shake at 4 °C for 24 h, wash with water three times, and then continue to shake with 10 mg / mL of PAH for 30 min. After centrifugation and washing, drug-loaded nanoparticles are obtained.

[0048] Specifically, the mass ratio of KMnO4 to sSiO2 is 15:2, and the mass ratio of PAH to hMnO2 is 2:1.

[0049] Specifically, Step 2 is as follows: Add a 1 mg / mL LOX solution to the nanocarrier, shake at 4 °C for 30 min, then add a 1 mg / mL vaccine protein solution, vortex to mix evenly, and repeatedly extrude the mixed solution 11 times with a liposome extruder.

[0050] In the present invention, the filter membrane diameter of the liposome extruder is 400 nm.

[0051] Specifically, the feeding mass ratio of hMnO2 to LOX and vaccine protein is 10:1 and 1:1.

[0052] Example 1, Preparation of lactate-responsive nanocomposite (TMLV):

[0053] 1. Prepare a positively charged hMnO2 nano-loading system:

[0054] The hMnO2 nanoparticles are hollow mesoporous nanocarriers prepared by the template etching method. The template etching method is specifically as follows: Slowly add a 10 mg / mL KMnO4 solution to an aqueous solution of 1 mg / mL sSiO2 nanoparticles, and react under ultrasonic conditions for 6 h. After centrifuging (12,000 rpm, 15 min) to collect the product, etch the sSiO2 component in the product with a 2 M Na2CO3 solution to obtain the hMnO2 nanoparticles.

[0055] Subsequently, disperse 10 mg of hMnO2 in a 5 mg / mL TPZ solution, shake at 4 °C for 24 h, wash with water, and then continue to shake with 10 mg / mL of polyallylamine hydrochloride (PAH) for 30 min. After centrifugation and washing, a positively charged hMnO2 nano-loading system is obtained.

[0056] 2. Load LOX and veterinary vaccine protein:

[0057] Add 0.5 mL of a 1 mg / mL LOX to the hMnO2 nanoparticles loaded with TPZ, quickly mix well, and then incubate in a constant temperature shaker at 4 °C for 30 min.

[0058] Subsequently, 10 mL of a 10 mg / mL inactivated veterinary canine coronavirus vaccine was added to the mixed solution. After mixing evenly, the mixed solution was repeatedly extruded 11 times using a liposome extruder to obtain the lactate-responsive nanocomposite (TMLV).

[0059] The size of the lactate-responsive nanocomposite (TMLV) was characterized using a transmission electron microscope and a dynamic light scattering instrument. The results are shown in Figure 1 a and Figure 1 b.

[0060] The results showed that the nanocomposite had an appropriate size and excellent dispersibility.

[0061] Meanwhile, the components in the nanocomposite were characterized using a UV-visible spectrophotometer and polyacrylamide gel electrophoresis. The results are shown in Figure 1 c and 1d. The results showed that all functional components were effectively combined in TMLV;

[0062] 3. Catalytic effect of the lactate-responsive nanocomposite (TMLV):

[0063] Lactate consumption: 50 mg / L of TMLV and TMV (without the LOX component) nanoparticles were respectively mixed evenly with a 10 mM lactate solution and oscillated at 37 °C. Solution samples were collected at preset time points, and the remaining lactate content in the solution was detected using a lactate detection kit.

[0064] Meanwhile, different concentrations of TMLV nanoparticles were respectively mixed evenly with a 10 mM lactate solution. After oscillating at 37 °C for 5 min, solution samples were collected, and the H2O2 content was detected using a hydrogen peroxide detection kit.

[0065] As shown in Figure 1 e-1f, the results showed that the TMLV nanomaterial could not only consume lactate but also produce a large amount of H2O2 for activating subsequent chemodynamic therapy.

[0066] Dissolved oxygen detection: TMV nanoparticles and different concentrations of TMLV nanoparticles were respectively added to a lactate solution sealed with paraffin liquid, and a dissolved oxygen meter was used to record the dissolved oxygen content in the solution.

[0067] The results are shown in Figure 1 g. TMLV consumed lactate while causing local hypoxia, which could be used to activate the chemotherapy effect of subsequent hypoxia drugs, further demonstrating that TMLV had lactate-activatable therapeutic properties.

[0068] Generation of reactive oxygen species: 100 mg / L of MLV (excluding TPZ), MV (excluding TPZ and LOX), and LOX + vaccine (veterinary vaccine protein) were respectively mixed evenly with the NaHCO3 / CO2 buffer solution. Subsequently, 2 μM of aminophenyl fluorescein was added to the above mixed solution, and 20 mM of lactate was selectively added. The production of ·OH in different solution systems was detected with a fluorescence spectrophotometer at the set time points.

[0069] The results are as Figure 1 shown in h. In the absence of H2O2, the MLV nanoparticles can generate ·OH through a catalytic cascade reaction. However, in the absence of lactate, almost no production of ·OH was detected in the MLV group, which further indicates that the TMLV nanoparticles have lactate-activatable chemodynamic therapy performance.

[0070] Example 2, anti-tumor effect in vitro:

[0071] First, mouse breast cancer (4T1) cells were evenly seeded in a 96-well plate (1×10 4 cells / well). After incubating with DMEM medium for 24 h, the TMLV active material and different control materials were co-cultured with the tumor cells respectively. After 12 h, the activity of the tumor cells was detected by the MTT method.

[0072] As Figure 2 shown in a- Figure 2 c, LOX and the vaccine protein itself do not show cytotoxicity. However, introducing the LOX component into other control materials can amplify the effects of chemotherapy and chemodynamic therapy, proving that the introduction of LOX can enhance the sensitivity of chemotherapy and chemodynamic therapy by catalyzing the oxidation of lactate. Among different treatment groups, TMLV showed the best cytotoxicity, indicating the effective synergy of chemotherapy and chemodynamic therapy, and TMLV showed lactate-activatable tumor-killing effects.

[0073] Example 3, effect of inducing immunogenic cell death:

[0074] Mouse breast cancer (4T1) cells were evenly seeded in a 96-well plate (2×10 5 cells / well). After incubating with DMEM medium for 24 h, the TMLV active material and different control materials were co-cultured with the tumor cells respectively. After 12 h, the cell supernatant was collected, and the content of HMGB1 in the supernatant was measured by enzyme-linked immunosorbent assay; meanwhile, the cells were collected, and the level of calreticulin on the cell membrane surface was detected by flow cytometry. As Figure 3 shown in a and 3b, the TMLV nanoparticles can significantly trigger the immunogenic cell death effect of tumors.

[0075] Example 4, effect of activating immune cells in vitro

[0076] In this example, the dendritic cells were extracted from C57BL / 6 mice.

[0077] Specifically, healthy C57BL / 6 mice were sacrificed by cervical dislocation, and the femurs of the mice were quickly dissected and placed in a sterile laminar flow hood. After soaking the femurs in disinfected alcohol for 5 min, bone marrow cells of the mice were aspirated using a sterile syringe and sterile forceps, and a single-cell suspension was obtained by filtering through a cell strainer. Subsequently, the cells were cultured in DMEM medium containing 20 ng / mL GM-CSF and 10 ng / mL IL-4, and the medium was changed every two days. Differentiated dendritic cells were obtained after 7 days. Then, macrophages and dendritic cells were evenly seeded in 6-well plates (2×10 5 cells / well) and treated with different materials. After 24 h, the cells were collected, stained with corresponding antibodies, and the activation of the two antigen-presenting cells was detected by flow cytometry.

[0078] The results are as Figure 4 shown. The nanocarrier material can effectively promote the activation of macrophages and the maturation of dendritic cells, and its immunomodulatory effect mainly comes from the veterinary vaccine components.

[0079] Example 5, in vivo immune effect:

[0080] In this example, the animal model was 7- to 8-week-old female BABL / c mice inoculated with subcutaneous 4T1 tumors.

[0081] Immunosuppression alleviation effect: When the tumor volume was approximately 100 mm 3 , the tumor-bearing mice were randomly divided into 6 groups, and on days 1 and 3, the mice were intravenously injected with TMLV nanomaterials and other control materials PBS, MLV (without TPZ), TLV (without hMnO2), TMV (without LOX), and TML (without vaccine protein). After 7 days, the tumor tissues of the mice were collected, fixed with paraformaldehyde, and the expression of CD206 in the tumor was evaluated by immunofluorescence staining experiments.

[0082] The results are as Figure 5 shown in a. Compared with other control groups, the expression of CD206 in the tumors of the MLV, TML, and TMLV groups was significantly downregulated compared with other groups, indicating that when LOX consumes lactic acid, it can effectively reduce the differentiation of M2 macrophages in the tumor and reverse immunosuppression.

[0083] Collection of single-cell suspension of mouse tumors: When the tumor volume was approximately 100 mm 3When the tumor-bearing mice were randomly divided into 6 groups, on the 1st and 3rd days, the mice were intravenously injected with TMLV nanomaterials and other control materials PBS, MLV, TLV, TMV, and TML. After 7 days, the tumor tissues of the mice were collected, and the tumor tissues were digested into single-cell suspensions with DMEM medium containing 0.1 mg / mL hyaluronidase, 0.1 mg / mL DNase, and 1 mg / mL collagenase. After filtering the above single-cell suspensions, they were washed twice with PBS containing 2% serum and divided into two portions and aliquoted into ep tubes (1×10 6 / tube). One portion of the tumor cells was stained with anti-CD11b and anti-CD80 antibodies. The other portion of the cells was stained with anti-CD3, anti-CD4, and anti-CD8 antibodies. After shaking at 4 °C for 25 min, the proportion of activated macrophages in the tumor and the infiltration levels of CD4 + T cells and CD8 + T cells were detected by flow cytometry respectively.

[0084] The activation of macrophages is shown in Figure 5 b. MLV, TMV, and TMLV groups all significantly activated the macrophages in the tumor, and the TMLV group had the best effect, indicating that the activation of macrophages mainly depends on the consumption of lactic acid and the stimulating effect of vaccine proteins.

[0085] Maturation of dendritic cells: When the tumor volume was approximately 100 mm 3 , the tumor-bearing mice were randomly divided into 6 groups, and on the 1st and 3rd days, the mice were intravenously injected with TMLV nanomaterials and other control materials PBS, MLV, TLV, TMV, and TML. After 7 days, the tumor-draining lymph node tissues of the mice were collected, and the lymph nodes were squeezed into single-cell suspensions with the plastic end of a syringe. The obtained cells were washed with PBS containing 2% serum and dispersed in ep tubes (1×10 6 cells, 100 μL), and appropriate amounts of anti-CD11c, anti-CD80, and anti-CD86 antibodies were added to stain the cells. Subsequently, the maturation ratio of dendritic cells was detected by flow cytometry.

[0086] The results are shown in Figure 5 c. Compared with other control groups, the TMLV nanocomposite showed the best promoting effect on the maturation of dendritic cells, indicating that the maturation of dendritic cells depends on the multiple immune regulatory effects mediated by TMLV, including the synergistic chemotherapy and chemodynamic therapy inducing a strong immunogenic death effect in tumor cells, and the inherent immune regulatory effect of vaccine proteins.

[0087] Example 6, in vivo treatment feasibility assessment:

[0088] Tumor enrichment effect: 4T1 cells (1×10 6 cells) were inoculated on the back of 7- to 8-week-old female BABL / c mice to construct 4T1 tumor-bearing mice. When the tumor volume of the mice was approximately 100 mm 3 , the tumor-bearing mice were intravenously injected with indocyanine green-labeled nanocomposites, and a near-infrared in vivo fluorescence imager was used to observe the enrichment of the nanocomposites in tumor tissues at different time points.

[0089] The results are as Figure 6 shown in a. The enrichment amount of the above nanocomposites reached the maximum at 12 h after injection at the tumor site, and had a good retention effect at the tumor site.

[0090] Biosafety assessment: 7- to 8-week-old healthy BABL / c mice were randomly divided into two groups, and were intravenously injected with TMLV nanocomposites and PBS, respectively. After 7 days, blood samples of the mice were collected by cardiac puncture, and the blood parameters of the mice were detected using blood biochemical and blood routine instruments.

[0091] The results are as Figure 6 shown in b and Figure 6 c. Compared with the blank control group, the blood parameters of the mice in the TMLV group did not change significantly, indicating that the TMLV nanocomposites have good biosafety.

[0092] Example 7, in vivo treatment effect:

[0093] The animal model involved in this example was 7- to 8-week-old female BABL / c mice inoculated with subcutaneous 4T1 tumors.

[0094] When the tumor volume was approximately 100 mm 3 , the tumor-bearing mice were randomly divided into 6 groups, and on the 1st and 3rd days, the mice were intravenously injected with TMLV nanomaterials and other control materials PBS, MLV, TLV, TMV, and TML. The tumor volume of the mice was recorded every day during the treatment. On the 15th day of the treatment, the tumor tissues of the mice were collected and subjected to pathological analysis by H&E staining.

[0095] The growth curve of the mice tumors during the treatment is as Figure 7 shown in a. Compared with the PBS group, the other control groups (MLV, TLV, TMV, and TML) inhibited the growth of tumors at the initial stage of the treatment, but had poor effects in the later stage of the treatment. In contrast, TMLV showed good tumor suppression effects throughout the treatment cycle, indicating that the chemotherapy, chemodynamic therapy, and immunotherapy effects based on TMLV synergy demonstrated excellent anti-tumor performance.

[0096] The highly efficient anti-tumor chemo-immunotherapy nanocomposite responsive to lactic acid provided by the present invention adopts a strategy of anti-tumor chemotherapy and chemodynamic therapy activated by lactic acid response, uses vaccine protein instead of traditional adjuvant, effectively overcomes the toxic and side effects in the treatment process, and can effectively activate the immune response of tumors by regulating multiple pathways.

[0097] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A lactate-responsive nano-composite for anti-tumor chemo-immunotherapy, characterized in that: The structure of the nanocomposite is a nanostructure formed by the complexation of PEGylated virus vaccine protein, lactate oxidase, and hollow manganese dioxide loaded with hypoxia-sensitive chemotherapeutic drugs.

2. The nano - composite material for lactate - responsive anti - tumor chemo - immunotherapy according to claim 1, wherein: The hypoxia-sensitive chemotherapeutic drug is loaded into the inner cavity of the hollow manganese dioxide in the nanocomposite.

3. The nano-composite material for lactate-responsive anti-tumor chemo-immunotherapy according to claim 1 or 2, characterized in that: The diameter of the nanocomposite is 150 nm - 200 nm.

4. A preparation method of a lactate-responsive anti-tumor chemo-immunotherapy nanocomposite, characterized in that, It includes the following steps: Step 1, prepare a positively charged hollow manganese dioxide nano-loading system: uniformly disperse hollow manganese dioxide nanoparticles in a chemotherapeutic drug solution, after oscillating and washing, complex with a positively charged polymer solution to obtain a positively charged nano-loading system; Step 2, load lactate oxidase and virus vaccine protein: add lactate oxidase to the nano-loading system, oscillate and then centrifuge, add PEGylated virus vaccine protein and mix evenly, and form a nanocomposite by repeated extrusion.

5. The preparation method of a lactate-responsive anti-tumor chemo-immunotherapy nanocomposite according to claim 4, characterized in that: The PEGylated virus vaccine protein is an inactivated veterinary canine coronavirus vaccine, the positively charged polymer solution is polyallylamine hydrochloride, and the chemotherapeutic drug is a hypoxia-responsive tumor chemotherapy drug.

6. The preparation method of a lactate-responsive anti-tumor chemo-immunotherapy nanocomposite according to claim 5, characterized in that: The hypoxia-responsive tumor chemotherapy drug is tirapazamine.

7. The preparation method of a lactate-responsive anti-tumor chemo-immunotherapy nanocomposite according to claim 4, characterized in that, Step 1 includes the following steps: Step 1.1: React a KMnO4 solution with an aqueous sSiO2 nanoparticle solution under ultrasonic conditions, after centrifuging to collect the product, etch the sSiO2 component in the product with a Na2CO3 solution to obtain hMnO2 nanoparticles; Step 1.2: Disperse hMnO2 in a chemotherapeutic drug solution, oscillate, wash with water, continue to oscillate with a positively charged polymer solution, and obtain drug-loaded nanoparticles after centrifugal washing.

8. The preparation method of a lactate-responsive anti-tumor chemo-immunotherapy nanocomposite according to claim 7, characterized in that: The mass ratio of KMnO4 to sSiO2 is 15:2, and the mass ratio of the positively charged polymer solution to hMnO2 is 2:

1.

9. The preparation method of a lactic acid-responsive anti-tumor chemo-immunotherapy nanocomposite according to claim 4, wherein, Specifically, Step 2 is: add a lactate oxidase solution to the nano-loading system, oscillate, add a vaccine protein solution, vortex and mix evenly, and repeatedly extrude the mixed solution with a liposome extruder.

10. The preparation method of a lactate-responsive anti-tumor chemo-immunotherapy nanocomposite according to claim 9, characterized in that: The feeding mass ratio of hollow manganese dioxide to lactate oxidase and vaccine protein is 10:1 and 1:1.

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